Sleeping Beauty transposon system for mutagenesis in zebrafish

描述（由申请人提供）：自1993年以来，美国的癌症死亡率稳步下降，但总体下降幅度相对较小，部分原因是侵袭性和转移性疾病的治疗策略不足。这部分源于我们对促进癌细胞转移行为的遗传变化的不完全理解。缺乏这种知识阻碍了新疗法的发展，以改变细胞通路导致转移。我们的长期目标是确定驱动肿瘤诱导和转移的遗传途径，以揭示新的治疗靶点。本文的目的是建立一个诱变系统，将确定在肿瘤进展到转移的基因合作。我们先前在斑马鱼中开发了一种稳健的转座子体细胞诱变策略，该策略导致成体组织中的肿瘤诱导和癌症基因的鉴定。我们建议扩大我们原来的系统，使其适用于体细胞和生殖系诱变。将产生诱导型和生殖系靶向转座酶来源，用于与现有Gal4驱动系一起使用。我们设计了一种新的基因断裂转座子，含有荧光报告捕获编码和非编码基因的表达。荧光报告基因将允许在移植到wil型受体后的肿瘤发生期间跟踪单细胞。我们将结合我们最近在实验室分离的视网膜肿瘤模型来测试我们的突变系统促进转移行为的能力。转座子整合位点分析将在表现出转移行为的单细胞上进行。目标是确定与单个癌细胞转移行为相关的基因通路。我们提出的工作的基本原理是，我们的诱变系统与成年和儿童癌症的斑马鱼模型相结合，将鉴定在肿瘤发病和转移中合作的新基因。这些信息有望揭示治疗转移性疾病的新的治疗靶点。

DESCRIPTION (provided by applicant): The U.S. has seen a steady decline in cancer mortality rates since 1993, but the overall decrease is relatively slight, due in part to insufficint treatment strategies for invasive and metastatic disease. This stems in part from our incomplete understanding of the genetic changes that promote metastatic behavior in cancer cells. Lack of such knowledge hinders the development of new therapies to target altered cellular pathways leading to metastasis. Our long-term goal is to define genetic pathways that drive tumor induction and metastasis in order to reveal novel therapeutic targets. The objective here is to build a mutagenesis system that will identify genes that cooperate in tumor progression to metastasis. We previously developed a robust transposon somatic mutagenesis strategy in zebrafish that leads to tumor induction in adult tissues and identification of cancer genes. We propose to expand our original system so it is applicable to both somatic and germ line mutagenesis. Inducible and germ line targeted transposase sources will be created for use with existing Gal4 driver lines. We have designed a novel gene breaking transposon containing a fluorescent reporter to capture expression of coding and noncoding genes. The fluorescence reporter will allow single cells to be followed during tumorigenesis after transplantation into wil type recipients. We will test the ability of our mutagenesis system to promote metastatic behavior in combination with a retinal tumor model we recently isolated in our laboratory. Transposon integration site analysis will be carried out on single cells exhibiting metastatic behavior. The goal is to identify gene pathways that correlate with metastatic behavior of individual cancer cells. Our rationale for the pro- posed work is that our mutagenesis system in combination with zebrafish models of adult and childhood cancers will identify new genes that cooperate in tumor onset and metastasis. This information can be expected to reveal new therapeutic targets for treating metastatic diseases.